EP2907871B1

EP2907871B1 - Thermostable Type-A DNA polymerase mutant with increased resistance to inhibitors in blood

Info

Publication number: EP2907871B1
Application number: EP15154718.9A
Authority: EP
Inventors: Nancy Mckinney; Holly Hogrefe; Jeffrey Fox; Connie Hansen; Bahram Arezi
Original assignee: Agilent Technologies Inc
Current assignee: Agilent Technologies Inc
Priority date: 2014-02-14
Filing date: 2015-02-11
Publication date: 2018-09-19
Anticipated expiration: 2035-02-11
Also published as: US20150232821A1; CN104845950B; US9758773B2; JP6466192B2; EP2907871A1; CN104845950A; JP2015156858A

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No. 61/940,172 filed on February 14, 2014 .

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. More specifically, the invention relates to improved polymerases for performing Polymerase Chain Reactions (PCR) and to methods of performing PCR.

BACKGROUND OF THE INVENTION

PCR is one of the most widely used nucleic acid detection methods. It has various applications in the fields of research and molecular diagnosis of infectious diseases and genetic disorders. Among the many applications, blood-based PCR diagnoses are particularly in demand.
On the one hand, the blood is a vital part of the circulatory system for the human body. It includes many types of host cells, including normal cells, diseased cells (e.g., circulating tumor cells), and, if the host is a pregnant mother, fetal cells, as well as genetic materials from such cells, such as microRNAs. Indeed, due to the minimum invasive nature, blood-based PCR is advantageous in some situations such as foetal diagnosis using a maternal blood sample without the risk of abortion associated with conventional amniotic fluid tests. Under certain circumstances, the blood also contains pathogens, such as viruses and bacteria. Accordingly, blood-based diagnoses can provide an immediate picture of what is happening in the human body at any given time. On the other hand, blood is the source for various pharmaceutical and blood products (e.g., whole blood, plasma, antibodies, and stem cells) that are used to improve the quality of life and to save lives in a variety of traumatic or pathological situations. These therapeutic uses of blood, plasma, and other blood-derived materials require that donations of these materials be as free as possible from contamination of diseased cells (e.g., tumor cells) or pathogens (e.g., HIV, HBV, and HCV). See, e.g., US Applications 20130316925 , 20130157253 , 20120329061 , 20120070827 , 20120034614 , 20070281307 , and 20070105121 .
However, blood-based PCR diagnoses have been limited by a number of logistic and technical challenges. The challenges associated with nucleic acid diagnostics from biological samples are many folds. Complex biological samples, such as blood and cell lysates, have various components that can inhibit DNA polymerases used in PCR reactions. These components include hemoglobin, immunoglobulin G, lactoferrin, and proteases in blood. Although various procedures have been developed to purify samples before attempting PCR reactions, these steps are generally time-consuming, labor-intensive, and might not achieve the purification required for the subsequent PCR. In addition, nucleic acid-containing cells (e.g., white blood cells and fetal cells) account for only a small fraction of a blood sample, precious nucleic acid can be lost from the sample before the PCR reaction step.
When working with blood, one key point is that a blood sample should be collected in anticoagulants to prevent clotting since isolation of nucleic acids from clotted blood is not efficient and most of the cells will be lost in the clot. However, anticoagulants routinely used in blood sample collection, such as EDTA and heparin, interfere with or inhibit PCR. For example, heparin is highly negatively charged and will co-extract with DNA and thereby inhibit PCR reactions. See e.g., Garcia et al. J. Clin. Microbiol. April 2002 vol. 40 no. 4 1567-1568, and Yokota et al., Journal of Clinical Laboratory Analysis, Volume 13, .
Thus, there is a need for reagents, such as DNA polymerases, that are more resistant to the above-mentioned inhibitors and suitable for PCR reactions using blood samples.
WO 2011/014885 describes DNA polymerases with an increased rate of incorporation of nucleotides into nucleic acids undergoing polymerization.

SUMMARY OF INVENTION

The subject matter of the invention is as set out in the appended claims. In particular, one aspect of the invention relates to an isolated, mutant thermostable Type-A DNA polymerase that is at least 90, 95, 96, 97, 98 or 99% identical to the sequence of SEQ ID NO: 4, and contains five amino acid substitutions at residues G59, V155, L245, E507, and F749 of SEQ ID NO:4 or at residues corresponding to residues G59, V155, L245, E507, and F749 of SEQ ID NO:4; wherein said substitutions are G59W, V155I, L245M, E507K, and F749I of SEQ ID NO:4; wherein the mutant polymerase possesses (i) DNA polymerase activity and (ii) a higher resistance to polymerization activity inhibitor heparin than the wild-type DNA polymerase from which the mutant polymerase is derived, and wherein the mutant polymerase is free of mutation at residue 375 or 734 of SEQ ID NO: 4, or at a residue corresponding to residue 375 or 734 of SEQ ID NO: 4.
In a further aspect, the invention provides a composition comprising (i) the above mutant polymerase of the invention and (ii) one or more reagents selected from the group consisting of an aqueous buffer, a divalent metal, extension nucleotides, primers, a detergent, a detection agent, and a target nucleic acid.
In a further aspect, the invention provides a method of amplifying a target nucleic acid, the method comprising: providing a test sample suspected of containing the target nucleic acid; contacting the test sample with the above mutant polymerase of the invention, a primer that specifically binds to the target nucleic acid, and extension nucleotides to form a mixture, and incubating the mixture under conditions permitting extension of the primer by the polymerase using the sequence of the target nucleic acid as a template for incorporation of the extension nucleotides.
In a further aspect, the invention provides an isolated nucleic acid comprising a nucleotide sequence that encodes the above mutant polymerase of the invention.
In a further aspect, the invention provides a vector or a host cell comprising the above nucleic acid of the invention.
In another aspect, the invention provides a method of producing the above mutant polymerase of the invention, comprising culturing the above host cell of the invention in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cell or the medium.
In another aspect, the invention provides a kit for amplification of a target nucleic acid, the kit comprising (i) the above mutant polymerase of the invention and (ii) one or more reagents selected from the group consisting of an aqueous buffer, a divalent metal, an extension nucleotide, a primer, a probe, a detergent, a detection agent, a dye, a fluorescent molecule, an anticoagulant, and a cell lysis agent.
This invention relates to thermostable Type A DNA polymerase mutants that have increased resistance to the inhibitor heparin in whole blood. Further disclosed inhibitors include natural components of blood (hemoglobin in erythrocytes, lactoferrin in leukocytes) or plasma (immunoglobulin G), or added anticoagulants, such as EDTA.
Accordingly, in one aspect, the invention provides an isolated, mutant thermostable Type-A DNA polymerase.
The mutant polymerase comprises, consists essentially of, or consists of a first mutation at residue 507 of a wild-type Taq DNA polymerase (SEQ ID NO: 4) or at a residue corresponding to residue 507 of the wild-type Taq DNA polymerase in another thermostable Type-A DNA polymerase; and additional mutations at residues 59, 155, 245, and 749 of the wild-type Taq DNA polymerase, or at corresponding residues in the another thermostable Type-A DNA polymerase. The mutant polymerase possesses (i) DNA polymerase activity and (ii) a higher resistance to a polymerization activity inhibitor (e.g., natural components of blood or anticoagulants such as EDTA or heparin) than the wild-type DNA polymerase from which it is derived. The mutant polymerase is free of mutation at residue 375 or 734 of the wild-type Taq DNA polymerase, or at corresponding residues in the another thermostable Type-A DNA polymerase. The mutant can also possess a faster polymerization rate as compared with the wild-type DNA polymerase.
In some embodiments and disclosures, the wild-type Type-A DNA polymerase comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NOs: 1-10. The mutant DNA polymerase is at least 70% (e.g., 75, 80, 85, 90, 95, 96, 97, 98, or 99%) identical to a sequence selected from the group consisting of SEQ ID NOs: 1-10. In one example, the wild-type Taq DNA polymerase comprises, consists essentially of, or consists of the sequence of SEQ ID NO: 4. In other words, the mutant polymerase is a mutant Taq DNA polymerase.
In a preferred embodiment, the first mutation in the mutant is an E507K mutation based on the sequence of SEQ ID NO: 4. In a more preferred embodiment, the mutant contains the following five mutations: G59W, Y155I, L245M, E507K, and F749I. For example, the mutant can be one comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 11. In other embodiments and disclosures, the mutant can be a DNA polymerase that has the above-described five mutations and is at least 70% (e.g., 75, 80, 85, 90, 95, 96, 97, 98, or 99%) identical to the sequence of SEQ ID NO: 11.
The invention further provides a composition (e.g., a master mix for PCR reactions) comprising (i) the mutant DNA polymerase described above and (ii) one or more reagents selected from the group consisting of an aqueous buffer, a divalent metal (e.g., magnesium), extension nucleotides, primers, a detergent, a detection agent (e.g., specific or non-specific dyes or fluorescent molecules), and a target nucleic acid template. Also provided is a kit containing the mutant DNA polymerase or composition described above, and packaging materials therefor. The kit can include one or more reagents selected from the group consisting of an aqueous buffer, a divalent metal, an extension nucleotide, a primer, a probe, a detergent, a detection agent, a dye, a fluorescent molecule, an anticoagulant, and a cell lysis agent.
The above-described mutant DNA polymerase can be used in a method of primer-extending or a method of amplifying a target nucleic acid. The method includes the steps of: providing a test sample (e.g., a blood sample) suspected of containing the target nucleic acid; contacting the test sample with the mutant polymerase, a primer that specifically binds or hybridizes to a strand of the target nucleic acid, and extension nucleotides to form a mixture; and incubating the mixture under conditions permitting extension of the primer by the polymerase using the sequence of the target nucleic acid as a template for incorporation of the extension nucleotides. The method can be a method of PCR, and in that case, a second primer that specifically binds or hybridizes to the complement of the target nucleic acid strand mentioned above can be used. The test sample can be a blood sample, which can account for at least 1% (2%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) v/v of the mixture.
The above-described mutant DNA polymerases, compositions (e.g., master mixes for PCR reactions), and methods are particularly useful for blood sample-based PCR reactions. For example, they can be used for real-time PCR from whole blood spotted on Guthrie cards (with no anticoagulants) or from plasma, which is made from whole blood treated with anticoagulants by removing cells by centrifugation. In this application, the whole blood samples can be partially purified (by attachment to Guthrie cards or by centrifugation to remove red blood cells) to remove heme and other factors that quench florescence detection.
Another aspect of this invention provides an isolated nucleic acid that encodes the above-mentioned mutant DNA polymerase polypeptide. The nucleic acid can contain a sequence that is at least 70% (e.g., 80, 85, 90, 95, or 99%) identical to SEQ ID NO: 12. The invention also provides a vector, such as an expression vector, comprising the nucleic acid and a host cell comprising the nucleic acid. The nucleic acid, vector, and host cell can be used for producing a mutant DNA polymerase polypeptide of this invention. Accordingly, this invention also provides a method for producing the polypeptide. The method includes culturing the host cell in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cell or the medium of the cell.
The details of one or more embodiments of the invention are set forth in the description below. Other features, objectives, and advantages of the invention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of exemplary thermostable Type-A DNA polymerases that can be mutated to create an engineered DNA polymerase according to the invention.
FIG. 2 is a photograph showing amplification by Taq mutants 1C2 and 2C2 of a 322-bp fragment of the human IGF gene from DNA in PCR reactions containing 10-50% v/v heparin-treated whole blood.
FIGs. 3A-C are a set of photographs showing comparison of the activity of Taq mutant 1C2 with those of DNA polymerases from different sources in the presence of (A) 2.5% v/v EDTA-treated whole blood, (B) 25% v/v EDTA-treated whole blood, and (C) 45% v/v EDTA-treated whole blood.
FIGs. 4A-C are a set of photographs showing comparison of the activity of Taq mutant 1C2 with those of DNA polymerases from different sources in the presence of (A) 2.5% v/v heparin-treated whole blood, (B) 25% v/v heparin-treated whole blood, and (C) 45% v/v heparin-treated whole blood.
FIGs. 5A-F are a set of photographs showing amplification by six Taq mutants of the 322-bp fragment of the human IGF gene in PCR reactions containing 5-30% v/v heparin-treated whole blood.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, at least in part, on the unexpected discovery of certain mutant DNA polymerases that are more resistant to inhibitors present in blood samples than their wild-type counterparts.

1. Mutant DNA Polymerases

In a first aspect, the present invention provides genetically engineered or mutant, isolated DNA polymerases, which can be suitable for use in PCR reactions.
As disclosed herein, the engineered DNA polymerases of the invention are resistant to the DNA polymerase inhibitor heparin. More specifically, a DNA polymerase according to this aspect of the present invention comprises at least one mutation, as compared to the wild-type DNA polymerase from which it derives, that allows for acceptable levels of DNA polymerization or correct amplification of a desired product during PCR in the presence of heparin that reduces the polymerization rate of the wild-type DNA polymerase to a level that does not permit successful product formation in a PCR reaction. Any assay known in the art for determining polymerase activity and/or product formation can be used, for example, an assay as described in the examples below.
The DNA polymerases of this aspect of the invention typically have (i) a first mutation at residue 507 of SEQ ID NO: 4 (a wild-type Thermus aquaticus Taq DNA polymerase) or at a residue corresponding to residue 507 of the wild-type Taq DNA polymerase in another thermostable Type-A DNA polymerase; and (ii) additional mutations at residues 59, 155, 245, and 749 of the wild-type Taq DNA polymerase, or at corresponding residues in the another thermostable Type-A DNA polymerase. FIG. 1 lists ten examples of such a wild-type DNA polymerase and an alignment of their primary amino acid sequences. Residues corresponding to 59, 155, 245, 507, and 749 of SEQ ID NO: 4 in each polymerase can be located in FIG. 1. In exemplary embodiments, the following mutations are present in the Taq DNA polymerase, or at residues corresponding to these residues at G59W, Y155I, L245M, E507K, and F749I of SEQ ID NO: 4. Shown below are the amino acid sequence of one exemplary mutant, Taq 1C2, where the five mutations are underlined, and its coding sequence (SEQ ID NOs 11 and 12, respectively):

SEQ ID NO: 11
SEQ ID NO: 12

The DNA polymerases of this aspect of the invention are more resistant to blood treated with heparin than their wild-type counterparts. Disclosed DNA polymerases can also be more resistant to other inhibitors of Taq DNA polymerase. Examples of these other inhibitors include whole blood (with or without anticoagulants), fractions of whole blood (such as those collected using Guthrie card spots), or components of blood, such as blood plasma, hemoglobin, heme, immunoglobulin G, and lactoferrin; EDTA; cell lysates, such as ones containing inhibitory concentrations of polysaccharides; plant substances, such as pectin, xylan, and acidic polysaccharides; substances found in soil samples, such as humic acid, fulvic acid, and metal ions, including heavy metals and heavy metal ions; and certain organic solvents. Additional non-limiting examples of the inhibitors include urea, organic and phenolic compounds (e.g., phenol), glycogen, fats, calcium, cellulose, nitrocellulose, mineral oil, pollen, glove powder, SDS, and detergents. Various other inhibitors are known in the art, including without limitation those discussed in Kermekchiev, et al., "Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples", Nucleic Acids Research, Vol. 1, p. 14, 2008; and Abu Al-Soud, W., et al., "Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples", Applied and Environmental Microbiology, Vol. 64, No. 10, October 1998. As the analysis of blood samples from a subject is of importance in medical and forensic analyses, resistance to inhibitors found in blood or blood fractions, including inhibitors that are commonly added to blood to stabilize it and prevent coagulation (e.g., heparin) is a characteristic of the mutant enzymes according to this invention.
In preferred embodiments, an engineered DNA polymerase according to the invention possesses both increased resistance to the DNA polymerase inhibitor heparin and increased DNA polymerization rate as compared to their wild-type counterparts. Such polymerases thus are capable of polymerizing a nucleic acid strand from a primed DNA template at an increased rate even in the presence of heparin, which is widely known to be inhibitory to the polymerization rate of the wild-type DNA polymerase.
As used herein, the term "genetically engineered" is used interchangeably with "mutant" to indicate a protein or nucleic acid that has been altered in its sequence from the wild-type sequence to include an amino acid residue or nucleotide that is different from the corresponding residue or nucleotide in the wild-type protein or nucleic acid, respectively, from which it is derived. Mutants according to the invention thus include site-directed mutants in which specific residues have been intentionally changed, including deletions of one or more residues; insertions of one or more residues, and replacement of one or more residues of one Type-A DNA polymerase with an exogenous sequence, such as a corresponding sequence of another Type-A DNA polymerase. In situations where a replacement/substitution of one or very few residues is made to create a mutant, it is a straightforward matter to identify the DNA polymerase "from which the mutant is derived." However, in situations where regions of sequences are replaced by other regions of sequences, it is sufficient to understand that the mutant can be considered "derived" from either/any of the wild-type thermostable Type-A DNA polymerases from which sequences of the mutant show identity, particularly any one of the exemplary wild-type DNA polymerases shown below in FIG. 1 (SEQ ID NOs: 1-10).
In the exemplary embodiments discussed in detail herein, the DNA polymerases of the invention are mutant forms of wild-type Taq DNA polymerase, which have altered features that provide the mutant polymerases with advantageous properties. However, it is to be understood that the invention is not limited to the exemplary embodiments discussed in detail below. For example, the invention includes mutants of polymerases other than Taq DNA polymerase, such as mutants of any thermostable Type-A family DNA polymerase. These mutants can be mutants of the polymerases, including but not limited to those, from species of Thermus or Thermatoga. It is well documented and well understood by those of skill in the art that thermostable Type-A DNA polymerases show high levels of sequence identity and conservation. Thus, it is a simple matter for one of skill in the art to identify residues of one particular Type-A DNA polymerase that correspond to residues of another. Thus, reference herein to specific mutations in wild-type Taq DNA polymerase can easily be correlated to corresponding mutations in other polymerases.
FIG. 1 presents an alignment of the primary amino acid sequences of several non-limiting exemplary thermostable Type-A DNA polymerases. As shown in FIG. 1, various regions of thermostable Type-A DNA polymerases are highly conserved while other regions are variable. Those of skill in the art will immediately recognize and understand that mutations in addition to those specifically identified and discussed herein may be made in the variable regions of Type-A DNA polymerases without altering, or without substantially altering, the polymerase activity of the mutated enzyme. Likewise, conservative mutations at conserved residues may be made without altering, or substantially altering, the polymerase activity of the mutated enzyme. Mutating enzymes based on comparative structure analysis with other related enzymes is a common and useful technique in the molecular biology field that allows a person of skill to reasonably predict the effect of a given mutation on the enzymatic activity of the enzyme. Using the structural data and known physical properties of amino acids, those of skill in the art can mutate enzymes, such as the DNA polymerases encompassed by the present invention, without altering, or without substantially altering, the essential enzymatic characteristics of the enzymes.
As used herein, the term "isolated polypeptide" refers to a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide can constitute at least 10% (i.e., any percentage between 10% and 100% inclusive, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide of the invention can be produced by recombinant DNA techniques, or by chemical methods.
The "percent identity" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
The amino acid composition of the above-mentioned DNA polymerase peptide/polypeptide/protein may vary without disrupting the ability to catalyze the replication of DNA under primer extension reaction conditions and/or PCR reaction conditions as described herein in the presence of EDTA- or heparin-treated blood. For example, it can contain one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in the above-mentioned sequences, such as SEQ ID NO: 11, is preferably replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for the ability to catalyze the replication of DNA under PCR conditions described herein to identify mutants that retain the activity as descried below in, e.g., US 20110027833 .
While a variety of mutations can be made at each residue of the above-mentioned wild-type Thermus aquaticus Taq DNA polymerase (SEQ ID NO: 4) or corresponding residue of other wild-type DNA polymerase (e.g., SEQ ID NOs: 1-3 and 5-10) noted above, mention may be made of the following non-limiting mutations regarding the mutants disclosed herein, which have mutations at G59, V155, L245, E507, and F749 of SEQ ID NO: 4. In some embodiments, each residue at these positions is altered to another member of the respective families based on side chain similarity.
As those of skill in the art will immediately recognize, equivalent sequences of those of the exemplary SEQ ID NOs can be easily created by making one or more conservative substitutions at one or more residues not specified. Such equivalent sequences retain the essential polymerase characteristics of the mutant enzymes. The various conservative substitutions for different amino acids are discussed above and known in the art. Important regions and residues for DNA polymerase activity of Taq DNA polymerase and other Type-A DNA polymerases are well characterized, and those of skill in the art are well aware of which regions and residues can be altered without disrupting the activity of the DNA polymerase of interest. See, e.g., Alba, Replicative DNA polymerases, Genome Biol. 2001; 2(1): reviews3002.1-reviews3002.4 and Steitz, DNA Polymerases: Structural Diversity and Common Mechanisms. June 18, 1999 The Journal of Biological Chemistry, 274, 17395-17398.
An exemplary comparison of selected thermostable Type-A polymerases is provided in FIG. 1 to give the reader an understanding of conserved and variable regions within this group of enzymes; however, those of skill in the art will be aware of other alterations that can be made without substantially altering the activities discussed herein. In view of the fact that production of recombinant proteins is a routine matter in the field of biotechnology today, and as polymerase assays, such as Taq DNA polymerase assays, are well known and widely practiced as routine assays, production of mutant polymerases according to the present invention using the information provided herein is a routine matter for those of skill in the art. Automation and very powerful techniques and kits allow those practicing the invention to rapidly and routinely identify mutants according to the invention, and identify the particular levels of polymerase activity of interest (i.e., polymerization rate and polymerization rate in the presence of heparin).
The invention also provides a functional equivalent of a peptide, polypeptide, or protein of this invention, which refers to a polypeptide derivative of the peptide, polypeptide, or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It retains substantially the activity of the above-mentioned DNA polymerase under PCR conditions described herein (e.g., a PCR reaction mixture containing a blood sample that has been pre-treated with heparin and accounts for at least 1% (e.g., 2%, 2.5%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) v/v of the mixture.). The isolated polypeptide can contain SEQ ID NO: 11 or a functional fragment or equivalent thereof. In general, the functional equivalent is at least 70% (e.g., any number between 70% and 100%, inclusive, e.g., 70 %, 75%, 80%, 85%, 90%, 95%, and 99%) identical to SEQ ID NO: 11.
A polypeptide described in this invention can be obtained as a recombinant polypeptide. To prepare a recombinant polypeptide, a nucleic acid encoding it can be linked to another nucleic acid encoding a fusion partner, e.g., glutathione-s-transferase (GST), 6x-His epitope tag, or M13 Gene 3 protein. The resultant fusion nucleic acid expresses in suitable host cells a fusion protein that can be isolated by methods known in the art. The isolated fusion protein can be further treated, e.g., by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of this invention. Alternatively, the peptides/polypeptides/proteins of the invention can be chemically synthesized (see e.g., Creighton, "Proteins: Structures and Molecular Principles," W.H. Freeman & Co., NY, 1983), or produced by recombinant DNA technology as described herein. For additional guidance, skilled artisans may consult Frederick M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003; and Sambrook et al., Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001).
The mutant DNA polymerases of the present invention can be provided in purified or isolated form, or can be part of a composition. Preferably, where in a composition, the mutant DNA polymerases are first purified to some extent, more preferably to a high level of purity (e.g., about 80%, 90%, 95%, or 99% or higher). Compositions according to the invention can be any type of composition desired, but typically are aqueous compositions suitable for use as, or inclusion in, a composition for amplification of a target nucleic acid, and in particular for blood-based amplification, such as through use of a PCR technique. As such, the compositions typically comprise at least one substance other than the mutant DNA polymerase, such as water, glycerol or another stabilizing agent, an aqueous buffer, an aqueous salt buffer, a divalent metal (e.g., magnesium) and the like. In exemplary embodiments, the compositions comprise some or all of the solvents, salts, buffers, nucleotides, and other reagents typically present in a PCR reaction. Thus, in some embodiments, the compositions comprise a magnesium salt (e.g., magnesium chloride or magnesium sulfate), one or more nucleoside triphosphates, one or more nucleic acid primers or probes, one or more additional nucleic acid polymerases or fragments thereof having desired activities, one or more polymerization detection agents (e.g., specific or non-specific dyes or fluorescent molecules), and/or one or more nucleic acid templates for amplification or sequencing. Other exemplary substances include detergents, DMSO, DMF, gelatin, glycerol, betaine, spermidine, T4 gene 32 protein, E. coli SSB, BSA, and ammonium sulfate. Those of skill in the art are well aware of the various substances that can be included in polymerization reaction compositions, and as such an exhaustive list is not necessary here.

2. Nucleic Acids, Vectors, and Host Cells

The present invention also provides a nucleic acid that encodes any of the mutant DNA polymerase polypeptides mentioned above. Preferably, the nucleic acid is isolated and/or purified. A nucleic acid refers to a DNA molecule (for example, but not limited to, a cDNA or genomic DNA), an RNA molecule (for example, but not limited to, an mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded. An "isolated nucleic acid" is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.
The present invention also provides recombinant constructs or vectors having one or more of the nucleotide sequences described herein. Examples of the constructs include a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred embodiment, the construct further includes regulatory sequences, including a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press).
A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector can be capable of autonomous replication or integration into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The vector of this invention includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. A "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as inducible regulatory sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, transfected, or infected the level of expression of protein desired, and the like.
Examples of expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of or Simian virus 40 (SV40), bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, a nucleic acid sequence encoding one of the polypeptides described above can be inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are within the scope of those skilled in the art.
The expression vector can also contain a ribosome binding site for translation initiation, and a transcription terminator. The vector may include appropriate sequences for amplifying expression. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance in E. coli.
The vector containing the appropriate nucleic acid sequences as described above, as well as an appropriate promoter or control sequence, can be employed to transform, transfect, or infect an appropriate host to permit the host to express the polypeptides described above (e.g., SEQ ID NO: 11). Examples of suitable expression hosts include bacterial cells (e.g., E. coli, Streptomyces, Salmonella typhimurium), fungal cells (yeast), insect cells (e.g., Drosophila and Spodoptera frugiperda (Sf9)), animal cells (e.g., CHO, COS, and HEK 293), adenoviruses, and plant cells. The selection of an appropriate host is within the scope of those skilled in the art. In some embodiments, the present invention provides methods for producing the above mentioned polymerase polypeptides by transforming, transfecting, or infecting a host cell with an expression vector having a nucleotide sequence that encodes one of the polypeptides. The host cells are then cultured under a suitable condition, which allows for the expression of the polypeptide.

3. Methods and Uses

The mutant DNA polymerases of the invention are suitable for various uses, including primer extension, nucleic acid polymerization reactions, and others requiring DNA polymerases.
For example, the engineered mutant DNA polymerases can be used in methods of primer extension or polymerizing nucleic acids from a primer or set of primers and a nucleic acid template. In general, the methods comprise: (A) exposing (such as combining together, mixing, contacting etc.) an engineered DNA polymerase according to the invention to (1) a target nucleic acid and (2) at least one primer suitable for priming polymerization of a nucleic acid that is complementary to one strand of the target nucleic acid, and (B) exposing (such as subjecting) the polymerase, target nucleic acid, and primer(s) to conditions that allow polymerization of nucleic acids from the primer(s). The step of exposing the polymerase to the other substances can be any action that results in exposure of the recited substances to each other such that they can physically interact. It thus can comprise adding the substances together in a composition, mixing the substances together in a composition (i.e., a mixture), etc. Exposing may be performed fully or partially manually, or fully or partially automatically (i.e., by way of machinery, robotics, etc.). As those of skill in the art are aware, a wide variety of nucleic acids can be subjected to copying, amplifying, sequencing, etc. Thus, the invention is not limited by the target nucleic acid, its sequence, length, etc. Further, those of skill in the art are fully aware of the parameters to be considered when designing primers for priming polymerization of a nucleic acid based on a target nucleic acid template. Thus, the invention is not limited by the identity or sequence of the primers. It is to be understood that, where amplification is desired (e.g., PCR), two primers having different sequences and having specificity for two different sequences on opposite strands of the target nucleic acid should be used. In addition, the step of exposing the combined substances to conditions that allow for polymerization can be any action that allows for polymerization. Many conditions suitable for polymerization are known in the art, and those of skill in the art may select any appropriate conditions, as the situation requires, without undue or excessive experimentation. Parameters to be considered include, but are not necessarily limited to, salt concentration, metal ion or chelator concentration, buffer concentration and identity, presence or absence of detergents and organic solvents, concentration of polymerase or other enzymes, presence or concentration of nucleotides or modified nucleotides, presence or concentration of polymerization inhibitors or terminators, presence or concentration of probes or dyes for detection of polymerization products, temperature, and length of time of exposure. In exemplary embodiments, the conditions that allow polymerization of nucleic acids from the primer(s) are conditions for a PCR reaction. As will be recognized by those of skill in the art, the step of exposing the substances to conditions for polymerization can be considered as a step of polymerizing, such as a step of amplifying a nucleic acid template.
The polymerases are advantageously used in any variation or type of PCR reaction for amplification of nucleic acids, including both DNA and RNA amplifications. For amplification of RNA templates (e.g., mRNAs or microRNAs), an RNA-dependent DNA polymerase (e.g., a reverse transcriptase; RT) can be used to make a DNA strand complementary to the RNA template, and a DNA polymerase of the invention can be used to amplify the DNA complementary strand.
In some exemplary embodiments, the PCR method is conducted on "dirty" samples, such as blood. In general, as used herein, a "dirty" sample is one that includes undefined substances, typically present originally in the environment where the target nucleic acid was present. Thus, a dirty sample in general is a sample in which the target nucleic acid was not purified prior to inclusion in the polymerization reaction.
Accordingly, the polymerase mutants and related methods can be used in various ways. For examples, they can be used for detecting blood infections at an early stage of infection with a pathogen and more particularly for detecting pathogens at low concentrations in circulation from a volume of blood from a subject (e.g., a patient) or in a biological product. Examples of such pathogens include fungi, bacteria (e.g., Mycobacterium tuberculosis and H. pylori) and viruses (e.g., HIV, HBV, and HCV). They also can be used for detecting genetic mutations, markers or polymorphisms (e.g., Single Nucleotide Polymorphism) associated with cancer or genetic disorders. Furthermore, they can be used for analysis of rare cells, such as foetal cells in a maternal blood sample or micrometastatic tumor cells, the detection of which is often complicated by the low abundance of such cells and because the biological sample often will comprise a majority of other cells or tissue material besides the rare cell of interest. The ability to analyze rare cells and other biological material of limited availability allows the development of new diagnostic methods with a less invasive character. See, e.g., US Applications 20130316925 , 20130157253 , 20120329061 , 20120070827 , 20120034614 , 20070281307 , 20070105121 , 20050009108 , and 20020155519 .
Due to their increased polymerization rate, the polymerase enzymes of the present invention are also well suited for "fast PCR" reactions, such as those described in, e.g., US20110027833 . Further, due to their resistance to inhibitors found in blood and blood products, disclosed and inventive polymerase enzymes are particularly well suited for "fast PCR" reactions in samples that contain blood or fractions of blood. Preferably, polymerase enzymes of the present invention are well suited for "fast PCR" reactions in "dirty" samples, such as those containing blood or fractions of blood that have been treated with the anticoagulant heparin.
In some embodiments, two or more primers having different sequences are used in the method. For example, in some embodiments two primers are used, where one primer specifically binds to one strand of the template DNA and the other binds to the other strand of the template DNA, allowing for production of a double-stranded polymerization product. In some embodiments, one primer is specific for a sequence present on a single-stranded RNA template, such as an mRNA. Polymerization of a first complementary strand of the RNA from the first primer provides a template for the second primer. Subsequent to a first polymerization, the first primer can prime polymerization from either the template RNA or the DNA complement. One or more nucleic acid probes having sequence specificity for the target nucleic acid (including a complementary strand of the target, where the target is single-stranded) can be included in the method to provide a means for detection.
Many PCR methods include probes, dyes, or other substances that allow for detection of polymerization (e.g., amplification) products. One example of such methods is Real-Time PCR. Accordingly, the method can include a step of including in the polymerization reaction a substance that allows for detection of polymerization products. Furthermore, the method of the invention encompasses methods that include one or more control reactions to determine if the methods, or particular method steps, have been performed successfully. The control reactions can be positive control reactions or negative control reactions. Those of skill in the art are fully capable of devising appropriate control reaction conditions without the need for particular steps to be detailed herein.

4. Kits

The invention encompasses kits and diagnostic systems for nucleic acid copying, primer extension, or amplification for detecting a target sequence. To that end, one or more of the reaction components for the methods disclosed herein can be supplied in the form of a kit for use in the detection of a target nucleic acid. In such a kit, an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate (e.g., by electrostatic interactions or covalent bonding).
The kit described herein includes one or more of the mutant DNA polymerases described above. The kit can include one or more containers containing one or more mutant DNA polymerases of the invention. A kit can contain a single mutant polymerase in a single container, multiple containers containing the same mutant DNA polymerase, a single container containing two or more different mutant DNA polymerases of the invention, or multiple containers containing different mutant DNA polymerases or containing mixtures of two or more mutant DNA polymerases. Any combination or permutation of DNA polymerase(s) and containers is encompassed by the kits of the invention. In a preferred embodiment, the kit includes 1)3 separate tubes containing a polymerase, a buffer (with Mg²⁺), and nucleotides or 2) a master mix containing a mixture of polymerase, buffer components, Mg²⁺, and nucleotides. Users of the kit can add suitable nucleic acid templates or test samples containing such templates (e.g., blood) and primers depending on their purposes and assay designs. The polymerase and/or buffer (or master mix) can further contain one or more detergents, and may also contain hot start antibody. Master mixes for real time PCR could additionally contain dye.
The kit may also contain additional materials for practicing the above-described methods. In some embodiments, the kit contains some or all of the reagents, materials for performing a method that uses a mutant DNA polymerase according to the invention. The kit thus may comprise some or all of the reagents for performing a PCR reaction using the DNA polymerase of the invention. Some or all of the components of the kits can be provided in containers separate from the container(s) containing the polymerases of the invention. Examples of additional components of the kits include, but are not limited to, one or more different polymerases, one or more primers that are specific for a control nucleic acid or for a target nucleic acid, one or more probes that are specific for a control nucleic acid or for a target nucleic acid, buffers for polymerization reactions (in IX or concentrated forms), magnesium, nucleotides and one or more dyes or one or more fluorescent molecules for detecting polymerization products. The kit may also include one or more of the following components: supports, terminating, modifying or digestion reagents, osmolytes, and an apparatus for detecting a detection probe.
The reaction components used in an amplification and/or detection process may be provided in a variety of forms. For example, the components (e.g., enzymes, nucleotide triphosphates, probes and/or primers) can be suspended in an aqueous solution or as a freeze-dried or lyophilized powder, pellet, or bead. In the latter case, the components, when reconstituted, form a complete mixture of components for use in an assay.
A kit or system may contain, in an amount sufficient for at least one assay, any combination of the components described herein, and may further include instructions recorded in a tangible form for use of the components. In some applications, one or more reaction components may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers. With such an arrangement, the sample to be tested for the presence of a target nucleic acid can be added to the individual tubes and amplification carried out directly. The amount of a component supplied in the kit can be any appropriate amount, and may depend on the target market to which the product is directed. General guidelines for determining appropriate amounts may be found in, for example, Joseph Sambrook and David W. Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001; and Frederick M. Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, 2003.
The kits of the invention can comprise any number of additional reagents or substances that are useful for practicing a method of the invention. Such substances include, but are not limited to: anticoagulants (e.g., EDTA and heparin), reagents (including buffers) for lysis of cells, divalent cation chelating agents or other agents that inhibit unwanted nucleases, control DNA for use in ensuring that the polymerase and other components of reactions are functioning properly, DNA fragmenting reagents (including buffers), amplification reaction reagents (including buffers), and wash solutions. The kits of the invention can be provided at any temperature. For example, for storage of kits containing protein components or complexes thereof in a liquid, it is preferred that they are provided and maintained below 0°C, preferably at or below -20°C, or otherwise in a frozen state.
The container(s) in which the components are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, bottles, or integral testing devices, such as fluidic devices, cartridges, lateral flow, or other similar devices. The kits can include either labeled or unlabeled nucleic acid probes for use in amplification or detection of target nucleic acids. In some embodiments, the kits can further include instructions to use the components in any of the methods described herein, e.g., a method using a crude matrix without nucleic acid extraction and/or purification.
The kits can also include packaging materials for holding the container or combination of containers. Typical packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like).
As used herein, a DNA polymerase that is "resistant to an inhibitor," refers to a DNA polymerase mutant or variant that allows for acceptable levels of DNA polymerization or/and correct amplification of a desired product during PCR in the presence of such an inhibitor that reduces the polymerization rate of the wild-type DNA polymerase to a level that does not permit successful product formation in a PCR reaction. In certain embodiments, the inhibitor is whole blood treated with heparin. And, the term "resistant to an inhibitor" also refers to the situations where the DNA polymerase mutant or variant allows for acceptable levels of DNA polymerization or/and correct amplification of a desired product from a PCR reaction mixture containing a blood sample that has been pre-treated with heparin and accounts for at least 1% (e.g., 2%, 2.5%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) v/v of the mixture.
As used herein, the term "target nucleic acid" or "target" refers to a nucleic acid containing a target nucleic acid sequence. A target nucleic acid may be single-stranded or double-stranded, and often is DNA, RNA, a derivative of DNA or RNA, or a combination thereof. A "target nucleic acid sequence," "target sequence" or "target region" means a specific sequence comprising all or part of the sequence of a single-stranded nucleic acid. A target sequence may be within a nucleic acid template, which may be any form of single-stranded or double-stranded nucleic acid. A template may be a purified or isolated nucleic acid, or may be non-purified or non-isolated.
As used herein the term "amplification" and its variants includes any process for producing multiple copies or complements of at least some portion of a polynucleotide, the polynucleotide typically being referred to as a "template." The template polynucleotide can be single stranded or double stranded. Amplification of a given template can result in the generation of a population of polynucleotide amplification products, collectively referred to as an "amplicon." The polynucleotides of the amplicon can be single stranded or double stranded, or a mixture of both. Typically, the template will include a target sequence, and the resulting amplicon will include polynucleotides having a sequence that is either substantially identical or substantially complementary to the target sequence. In some embodiments, the polynucleotides of a particular amplicon are substantially identical, or substantially complementary, to each other; alternatively, in some embodiments the polynucleotides within a given amplicon can have nucleotide sequences that vary from each other. Amplification can proceed in linear or exponential fashion, and can involve repeated and consecutive replications of a given template to form two or more amplification products. Some typical amplification reactions involve successive and repeated cycles of template-based nucleic acid synthesis, resulting in the formation of a plurality of daughter polynucleotides containing at least some portion of the nucleotide sequence of the template and sharing at least some degree of nucleotide sequence identity (or complementarity) with the template. In some embodiments, each instance of nucleic acid synthesis, which can be referred to as a "cycle" of amplification, includes creating free 3' end (e.g., by nicking one strand of a dsDNA) thereby generating a primer and primer extension steps; optionally, an additional denaturation step can also be included wherein the template is partially or completely denatured. In some embodiments, one round of amplification includes a given number of repetitions of a single cycle of amplification. For example, a round of amplification can include 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100 or more repetitions of a particular cycle. In one exemplary embodiment, amplification includes any reaction wherein a particular polynucleotide template is subjected to two consecutive cycles of nucleic acid synthesis. The synthesis can include template-dependent nucleic acid synthesis.
The term "primer" or "primer oligonucleotide" refers to a strand of nucleic acid or an oligonucleotide capable of hybridizing to a template nucleic acid and acting as the initiation point for incorporating extension nucleotides according to the composition of the template nucleic acid for nucleic acid synthesis. "Extension nucleotides" refer to any nucleotide capable of being incorporated into an extension product during amplification, i.e., DNA, RNA, or a derivative if DNA or RNA, which may include a label.
"Hybridization" or "hybridize" or "anneal" refers to the ability of completely or partially complementary nucleic acid strands to come together under specified hybridization conditions (e.g., stringent hybridization conditions) in a parallel or preferably antiparallel orientation to form a stable double-stranded structure or region (sometimes called a "hybrid") in which the two constituent strands are joined by hydrogen bonds. Although hydrogen bonds typically form between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), other base pairs may form (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).
The term "stringent hybridization conditions" or "stringent conditions" means conditions in which a probe or oligomer hybridizes specifically to its intended target nucleic acid sequence and not to another sequence. Stringent conditions may vary depending on well-known factors, e.g., GC content and sequence length, and may be predicted or determined empirically using standard methods well known to one of ordinary skill in molecular biology (e.g., Sambrook, J. et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Ch. 11, pp. 11.47-11.57, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).
As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
The term "about" generally refers to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean from 0.9-1.1. Other meanings of "about" may be apparent from the context, such as rounding off, so, for example "about 1" may also mean from 0.5 to 1.4.
As used herein, the term "subject" refers to any organism having a genome, preferably, a living animal, e.g., a mammal, which has been the object of diagnosis, treatment, observation or experiment. Examples of a subject can be a human, a livestock animal (beef and dairy cattle, sheep, poultry, swine, etc.), or a companion animal (dogs, cats, horses, etc).
The term "biological sample" refers to a sample obtained from an organism (e.g., patient) or from components (e.g., cells) of an organism. The sample may be of any biological tissue, cell(s) or fluid. The sample may be a "clinical sample" which is a sample derived from a subject, such as a human patient or veterinary subject. Such samples include, but are not limited to, saliva, sputum, blood, blood cells (e.g., white cells), amniotic fluid, plasma, semen, bone marrow, and tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. A biological sample may also be referred to as a "patient sample." A biological sample may also include a substantially purified or isolated protein, membrane preparation, or cell culture.
As used herein, the term "contacting" and its variants, when used in reference to any set of components, includes any process whereby the components to be contacted are mixed into same mixture (for example, are added into the same compartment or solution), and does not necessarily require actual physical contact between the recited components. The recited components can be contacted in any order or any combination (or subcombination), and can include situations where one or some of the recited components are subsequently removed from the mixture, optionally prior to addition of other recited components. For example, "contacting A with B and C" includes any and all of the following situations: (i) A is mixed with C, then B is added to the mixture; (ii) A and B are mixed into a mixture; B is removed from the mixture, and then C is added to the mixture; and (iii) A is added to a mixture of B and C. "Contacting a template with a reaction mixture" includes any or all of the following situations: (i) the template is contacted with a first component of the reaction mixture to create a mixture; then other components of the reaction mixture are added in any order or combination to the mixture; and (ii) the reaction mixture is fully formed prior to mixture with the template.
The term "mixture" as used herein, refers to a combination of elements, that are interspersed and not in any particular order. A mixture is heterogeneous and not spatially separable into its different constituents. Examples of mixtures of elements include a number of different elements that are dissolved in the same aqueous solution, or a number of different elements attached to a solid support at random or in no particular order in which the different elements are not spatially distinct. In other words, a mixture is not addressable. To be specific, an array of surface-bound oligonucleotides, as is commonly known in the art and described below, is not a mixture of surface-bound oligonucleotides because the species of surface-bound oligonucleotides are spatially distinct and the array is addressable.

EXAMPLE 1 Generation and Screening of Mutant Polymerases

Mutant Taq DNA polymerases were generated by random mutagenesis of a nucleic acid comprising the sequence of SEQ ID NO: 4, which encodes a wild-type Thermus aquaticus Taq DNA polymerase in the manner described in US 20110027833 .
Briefly, random mutant libraries were subjected to 5 rounds of selection under fast cycling conditions, followed by screening (after rounds 2, 4, and 5) to identify clones that support amplification using shortened extension times. Polymerases showing improved performance during Real-Time PCR under fast cycling conditions, as compared to wild-type Taq DNA polymerase, were subjected to DNA sequencing to identify mutations. Mutations of interest appearing in fast-amplifying clones were identified and recombined using site-directed mutagenesis. Recombinants were screened using Real-Time PCR with fast cycling conditions. Mutant polymerases that outperformed both wild-type Taq and the best performers from the original selection/screening were identified, sequenced, and purified for further characterization to identify clones with combinations of mutations that support PCR using the shortest extension times.
Selected mutant polymerases obtained in the manner described above were further characterized to assess their ability to polymerize a nucleic acid chain from a primer in the presence of blood treated with anticoagulants such as EDTA and heparin.

EXAMPLE 2 Characterization of Mutant Enzymes' Resistance to EDTA

In this example, assays were carried out to examine the ability of various mutant enzymes to amplify a target DNA during End-Point PCR in the presence of whole blood that had been collected and stored in EDTA, which was known to inhibit Taq DNA polymerase activity.

Specifically, PCR reactions containing components typical for End-Point assays were assembled using wild-type Taq (Taq2000, Agilent Technologies; SEQ ID NO: 4) and a number of mutants derived from it. The mutants examined include the above-mentioned mutant Taq 1C2, a number of mutants described in US 20110027833 , including Taq 42, Taq 3B, Taq 2C2, and Taq 5A2, and three other mutants Taq 7P, Taq 8P, and Taq 5. The table below summarizes the mutations in these mutants:

Enzyme	G59W	V155I	L245M	L375V	E507K	E734G	F749I	K508R
Taq 7B (WT)	_	_	_	_	_	_	_	_
Taq 42	+	_	+	+	+	+	+	+
Taq 2C2	+	+	+	+	+	+	+	_
Taq 1C2	+	+	+	_	+	_	+	_
Taq 3B	_	+	+	_	+	_	+	_
Taq 1	+	_	_	_	_	_	_	_
Taq 8P	_	+	_	_	_	_	_	_
Taq 5A2	_	_	_	_	+	_	_	_
Taq 7P	_	_	_	_	_	_	+	_
Taq 5	+	+	+	_	_	_	_	_

To examine these enzymes' resistance to EDTA, PCR was performed to amplify a 322 base-pair target of human IGF gene from human genomic DNA in whole blood that had been treated with EDTA. The EDTA-treated blood contains 1.8µg/µl K₂EDTA. In the PCR reactions, final concentrations of the blood ranged from 1% to 65% v/v. The human blood was added individually as the template to enzyme master mixes previously aliquotted into PCR strip tubes. Each polymerase-template combination was assayed in duplicate. Amplification was performed using 50 ng of each enzyme per 50 µl reaction. The thermocycling parameters were as follows: 95 °C for 5 minutes; 95 °C for 30 seconds; 60 °C for 30 seconds, and 72°C for 1 minute, for 30 cycles.
The amplification products were fractionated on an agarose gel pre-stained with ethidium bromide. The highest amount of blood that produced PCR product is summarized in the table below:

Enzyme EDTA-blood

Taq 7B (WT) <1%

Taq

42 60%

Taq 3B

50%

Taq 2C2 65%

Taq 5A2

45%

Taq

7P 15%

Taq 8P 15%

Taq 1C2 60%

Taq

1 15%
The results indicate that the wild-type Taq DNA polymerase was able to amplify the 322-bp product only when the EDTA-treated blood is less than 1% v/v of the reaction mixture. In contrast, many of the mutants were capable of producing specific target products in the presence of as high as more than 50% EDTA-treated blood. Among them, Taq 42, Taq 2C2, and Taq 1C2 are most resistant to EDTA, and they were capable of producing specific target products in the presence of 60%, 65%, and 60% EDTA-treated blood samples respectively.

EXAMPLE 3 Characterization of Mutant Enzymes' Resistance to Heparin

Heparin is another routinely used anticoagulant. In this example, assays were carried out to examine the abilities of the Taq 2C2 and Taq 1C2 mutant enzymes to amplify a target DNA in the presence of whole blood that had been treated with heparin. The heparinized blood contains 15.8USP units/ml sodium heparin.
PCR reactions containing components typical for End-Point assays were assembled and the assays carried out in the manner described above except that heparin-treated blood was used as the template for each reaction. In the PCR reactions, final concentrations of the heparin-treated blood ranged from 10% to 50% v/v. The results are shown in FIG. 2. As shown in FIG. 2, Taq 2C2 was able to produce a substantial amount of specific target product in the presence of 10-30% v/v heparin-treated blood. But, the amounts or yields of the target product generally decreased as the amount of heparin-treated blood increased. When the heparin-treated blood was 30% v/v or more, Taq 2C2 showed a rapid loss in activity and a total lack of activity in the presence of 50% v/v of heparin-treated blood. In contrast, Taq 1C2 consistently produced a substantial amount of specific products across the entire ranges tested (10% to 50% v/v). More surprisingly, Taq 1C2 kept almost the same activity level and produced an essentially equivalent amount of product in the presence of 10-50% v/v heparin-treated blood.
These results support that the Taq 1C2 mutant DNA polymerase possesses advantageous properties as compared to other EDTA-resistant mutant DNA polymerases described in Example 2.
Additional assays were further carried out to examine the abilities of the Taq 1C2, 2C2, 42, 5, 5A2, and 7P mutants to amplify the above-mentioned 322 base-pair target of human IGF gene from human genomic DNA in heparin-treated whole blood in the same manner on a SureCycler. The thermocycling parameters were as follows: 95 °C for 5 minutes followed by 30 cycles of 95°C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 1 minute. The reaction mixtures contained 300 nM each primer, a reaction buffer containing 15 mM Tris pH 8.8, 96 mM KCl, 2% DMSO, 2.5 mM MgCl₂, 200 µM for each of dA,dG, dC, and dT, 0.5 µl of the Taq mutant at 50 ng/l IN FDB with 0.5% each Igepal and Tween-20. After centrifugation to settle debris, 8 µl of each 50 µl reaction mixture was run on Nusieve TBE gels. The results are shown in FIG. 5.
Again, the Taq 1C2 mutant retained almost the same activity level and produced an essentially equivalent amount of product in the presence of 5-30% v/v heparin-treated blood. And, the Taq 1C2 mutant is the only one that amplified the target DNA in the presence of 30% v/v heparin-treated blood.

EXAMPLE 4 Comparison of Taq 1C2 with Commercially Available DNA Polymerases

In this example, the Taq 1C2 mutant DNA polymerase was further compared with a number of commercially available DNA polymerases. To that end, the target amplified from blood was a 232-bp non-coding single copy region of the human genome.
Briefly, whole blood samples were obtained from a subject and treated with either EDTA or heparin according to standard protocols. These samples were used as templates to amplify the 232-bp region. Each PCR reaction was 50ul volume and cycled 30X using the Agilent SureCycler (Agilent Technologies, Inc.); 400nM of each primer was used in all cases. The DNA polymerases used include the Taq 1C2 mutant ("Agilent") and polymerases marketed by a number of manufactures, including Kapa Biosystems Blood PCR Mix A ("KAPA-A," Kapa Biosystems), DNA Polymerase Technology Omni KlenTaq ("KlenTaq," Sigma-Aldrich Co. LLC), Clontech Terra Direct ("ClonTech," Clontech Laboratories, Inc.) and Thermo Scientific Phusion Blood Direct ("Thermo," Thermo Scientific). The reaction mixes were assembled according to manufacturers' instructions. Thermal cycling profiles comply with manufacturers' recommendations and are detailed below:

The amplification products were fractionated on a 4% Nusieve agarose gel pre-stained with ethidium bromide. The results are shown in FIGs. 3 and 4, where the sizing ladder was in 100bp increments, and the same amount of each product (8 µl or 6% of each 50 µl reaction) was loaded onto the gels. These DNA polymerases' resistance to EDTA or heparin are ranked below:

2.5% EDTA blood V/V:	Clontech > Agilent > Thermo > KAPA = KlenTaq
25% EDTA blood V/V:	Agilent = KlenTaq = Clontech> KAPA > Thermo
45% EDTA blood V/V:	Agilent > KAPA (KlenTaq, Clontech, Thermo: no visible product
2.5% heparin blood V/V:	KlenTaq > Clontech > Thermo > KAPA > Agilent
25% heparin blood V/V:	Clontech > Agilent = Thermo > KlenTaq (KAPA: no visible product)
45% heparin blood V/V:	Agilent >> Clontech, Thermo (KlenTaq, Thermo: no visible product)

As shown above, the Taq 1C2 mutant outperformed most of the other polymerases. In addition, it was found that the Taq 1C2 mutant was the only one showing good yields in the presence of 45% v/v of heparinized blood. The Taq 1C2 mutant was also the only one showing good resistance to both EDTA and heparin.
The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims.

SEQUENCE LISTING

<110> Agilent Technologies, Inc.
<120> Thermostable Type-A DNA Polymerase Mutant with Increased Resistance to Inhibitors in Blood
<130> EP98185 FZSZ298pau
<140> not yet assigned
<141> 2014-02-14
<150> 61/940,172
<151> 2014-02-14
<160> 12
<170> PatentIn version 3.5
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<213> Thermus thermophilus
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<213> Thermus caldophilus
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<210> 3
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<213> Thermus thermophilus
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<211> 832
<212> PRT
<213> Thermus aquaticus
<400> 4
<210> 5
<211> 833
<212> PRT
<213> Thermus scotoductus
<400> 5
<210> 6
<211> 831
<212> PRT
<213> Thermus flavus
<400> 6
<210> 7
<211> 830
<212> PRT
<213> Thermus oshimai
<400> 7
<210> 8
<211> 833
<212> PRT
<213> Thermus filiformis
<400> 8
<210> 9
<211> 893
<212> PRT
<213> Thermotoga maritima
<400> 9
<210> 10
<211> 893
<212> PRT
<213> Thermotoga neapolitana
<400> 10
<210> 11
<211> 832
<212> PRT
<213> Artificial
<220>
<223> Synthetic
<400> 11
<210> 12
<211> 2499
<212> DNA
<213> Artificial
<220>
<223> Synthetic
<400> 12

Claims

An isolated, mutant thermostable Type-A DNA polymerase that is at least 90, 95, 96, 97, 98 or 99% identical to the sequence of SEQ ID NO: 4, and contains five amino acid substitutions at residues G59, V155, L245, E507, and F749 of SEQ ID NO:4 or at residues corresponding to residues G59, V155, L245, E507, and F749 of SEQ ID NO:4;
wherein said substitutions are G59W, V155I, L245M, E507K, and F749I of SEQ ID NO:4;

wherein the mutant polymerase possesses (i) DNA polymerase activity and (ii) a higher resistance to polymerization activity inhibitor heparin than the wild-type DNA polymerase from which the mutant polymerase is derived, and

wherein the mutant polymerase is free of mutation at residue 375 or 734 of SEQ ID NO: 4, or at a residue corresponding to residue 375 or 734 of SEQ ID NO: 4.
A composition comprising (i) the mutant polymerase of claim 1 and (ii) one or more reagents selected from the group consisting of an aqueous buffer, a divalent metal, extension nucleotides, primers, a detergent, a detection agent, and a target nucleic acid.
A method of amplifying a target nucleic acid, the method comprising:
providing a test sample suspected of containing the target nucleic acid;

contacting the test sample with the mutant polymerase of claim 1, a primer that specifically binds to the target nucleic acid, and extension nucleotides to form a mixture, and

incubating the mixture under conditions permitting extension of the primer by the polymerase using the sequence of the target nucleic acid as a template for incorporation of the extension nucleotides.
The method of claim 3, wherein the method is a method of PCR.
The method of any one of claims 3-4, wherein the test sample is a blood sample.
The method of any one of claims 3-5, wherein the mixture contains at least 1% v/v of the blood sample.
An isolated nucleic acid comprising a nucleotide sequence that encodes the mutant polymerase of claim 1.
The nucleic acid of claim 7, wherein the nucleotide sequence is at least 70% identical to SEQ ID NO: 12.
A vector or a host cell comprising the nucleic acid of claim 7 or 8.
A method of producing a mutant polymerase of claim 1, comprising culturing the host cell of claim 9 in a medium under conditions permitting expression of a polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cell or the medium.
A kit for amplification of a target nucleic acid, the kit comprising (i) the mutant polymerase of claim 1 and (ii) one or more reagents selected from the group consisting of an aqueous buffer, a divalent metal, an extension nucleotide, a primer, a probe, a detergent, a detection agent, a dye, a fluorescent molecule, an anticoagulant, and a cell lysis agent.